Gene name - dorsal
Cytological map position - 36C2-9
Function - transcription factor
Symbol - dl
Genetic map position - 2-52.9
Classification - rel homolog
Cellular location - nuclear and cytoplasmic
|Recent literature||Crocker, J, Ilsley, G.R. and Stern, D.L. (2016). Quantitatively predictable control of Drosophila transcriptional enhancers in vivo with engineered transcription factors. Nat Genet [Epub ahead of print]. PubMed ID: 26854918
Genes are regulated by transcription factors that bind to regions of genomic DNA called enhancers. Considerable effort is focused on identifying transcription factor binding sites, with the goal of predicting gene expression from DNA sequence. Despite this effort, general, predictive models of enhancer function are currently lacking. This study combine quantitative models of enhancer function with manipulations using engineered transcription factors to examine the extent to which enhancer function can be controlled in a quantitatively predictable manner. These models, which incorporate few free parameters, can accurately predict the contributions of ectopic transcription factor inputs. The effect of individual transcription factors can be considered as independent submodules of activity that are combined in a linear manner to produce a sigmoidal output. Individual submodules can also encode more complex inputs. For example, a submodule is represented by the saturating cooperativity displayed by Dorsal and Twist, whose combined output has an upper limit. The models allow the predictable 'tuning' of enhancers, providing a framework for the quantitative control of enhancers with engineered transcription factors.
|Sandler, J. E. and Stathopoulos, A. (2016). Quantitative single-embryo profile of Drosophila genome activation and the dorsal-ventral patterning network. Genetics [Epub ahead of print]. PubMed ID: 26896327
During embryonic development of Drosophila melanogaster, the Maternal to Zygotic Transition (MZT) marks a significant and rapid turning point when zygotic transcription begins and control of development is transferred from maternally deposited transcripts. Characterizing the sequential activation of the genome during the MZT requires precise timing and a sensitive assay to measure changes in expression. This study used the NanoString nCounter instrument, which directly counts mRNA transcripts without reverse transcription or amplification, to study over 70 genes expressed along the dorsal-ventral (DV) axis of early Drosophila embryos, dividing the MZT into 10 time points. Transcripts were quantified for every gene studied at all time points, providing the first data set of absolute numbers of transcripts during Drosophila development. Gene expression was found to change quickly during the MZT, with early Nuclear Cycle (NC) 14 the most dynamic time for the embryo. twist is one of the most abundant genes in the entire embryo and mutants were used to quantitatively demonstrate how it cooperates with Dorsal to activate transcription and is responsible for some of the rapid changes in transcription observed during early NC14. Elements within the gene regulatory network were uncovered that maintain precise transcript levels for sets of genes that are spatiotemporally co-transcribed within the presumptive mesoderm or dorsal ectoderm. Using this new data, it was shown that a fine-scale, quantitative analysis of temporal gene expression can provide new insights into developmental biology by uncovering trends in gene networks including coregulation of target genes and specific temporal input by transcription factors.
Dorsal (DL) is the focal protein in the development of dorsoventral polarity in the developing fly. It is a transcription factor, activating and repressing zygotic genes responsible for differentiation along the dorsoventral axis during the early stages of development. Over the course of time measured in hours, even before cellularization, the rapidly dividing zygotic cells take up their positions at the periphery of the fertilized egg. Nuclei in the ventral portion stain positive for DL while nuclei in the dorsal region fail to stain. Nuclear staining is graded; nuclei closest to the ventral midline stain most strongly (Rushlow, 1989, Steward, 1989 and Roth, 1989).
The dorsal group comprises a whole class of genes acting in the mother and responsible for setting up the activation and nuclear transport of DL. Early in oogenesis the egg becomes polarized. Such polarization affects the follicle cells surrounding the egg. Ventral follicle cells take on a different developmental fate from dorsal follicle cells. For example, the release and activation of the Toll ligand Spätzle occurs only in the ventral region of the egg.
Once fertilization has taken place, Spätzle triggers signaling in the Toll receptor. The signals are transmitted via Tube and Pelle into the cytoplasm, resulting in the activation of Dorsal. Activation of Dorsal is held in abeyance by Cactus protein. Cactus binds Dorsal, preventing its nuclear transport. Signals from Toll result in the destruction of Cactus in ventral cells, whereupon Dorsal is released to continue its transportation into the nucleus.
The Cactus-Dorsal interactive system is preserved in vertebrates and used in activation of the immune system. The transcription factor NF kappa B is involved in the activation of immune system cells. The vertebrate homolog of Cactus, I kappa B (where I stands for inhibitor), keeps NF kappa B restrained in the cytoplasm until cell activation, at which time NF kappa B enters the nucleus. The adult fly uses the identical system in its immune system. Bacterial challenge causes the activation of Dorsal and a second Dorsal-like factor (Dif) in the fat body, an organ that serves the immune function in flies. The developing awareness of the importance of an immune response system in flies has initiated a new focus of interest in Dorsal research (Reichart, 1993 and Lemaitre, 1995).
Dorsal's nuclear function involves an interaction with transcription factor Bang senseless, here termed Dorsal switch protein 1 or DSP1 (FlyBase link: FBgn0000231) DSP1, an HMG-1/2-like protein, binds DNA in a highly cooperative manner with three members of the Rel family of transcriptional regulators (NF-kappaB, the p50 subunit of NF-kappaB, and the Rel domain of Dorsal). This cooperativity is apparent with DNA molecules bearing consensus Rel-protein-binding sites and is unaffected by the presence of a negative regulatory element, a sequence previously proposed to be important for mediating repression by these Rel proteins. The cooperativity observed in these DNA-binding assays is paralleled by interactions between protein pairs in the absence of DNA. In HeLa cells, as assayed by transient transfection, expression of DSP1 increases activation by Dorsal from the twist promoter and inhibits that activation from the zen promoter, consistent with the previously proposed idea that DSP1 can affect the action of Dorsal in a promoter-specific fashion (Brickman, 1999).
DSP1 has opposite effects on the activity of Dorsal assayed with regulatory sequences excised from the twist and zen promoters. These experiments were performed by transiently transfecting mammalian cells in culture. Thus, reporters containing either a 180-bp fragment from zen (a fragment sufficient to mediate repression in Drosophila) or the entire regulatory region of twist (from -1,438 to +38) were activated by cotransfection with DNA encoding Dorsal. Cotransfection with DNA encoding DSP1 has just the opposite effects on this Dorsal mediated activation of the two promoters: activation from the twist promoter is stimulated 4-fold, whereas that from the zen promoter is inhibited 3-fold. DSP1's stimulation of Dorsal-mediated activation from the twist promoter can be mapped to the defined enhancer elements or VARs. Thus, DSP1 also stimulates Dorsal-mediated activation if the template bears, instead of the intact twist promoter, a cassette that contains the two VARs that drive ventral-specific expression of the twist gene in the Drosophila embryo. The two VARs together constitute approximately 300 bp and contain multiple Rel-protein-binding sites (Brickman, 1999).
It is not known what DNA sequences in the zen and twist promoters determine the opposite effects of DSP1 on dorsal-mediated activation. The finding that a negative regulatory element (NRE) has no effect on cooperative binding to DNA of DSP1 and various Rel proteins prompted a reexamination of the earlier claims that DSP1 converts Dorsal, the p50 homodimer, and the NF-kappaB heterodimer into repressors and that this effect requires the NRE. In each case, DSP1 inhibits Rel-protein-dependent activation both in the presence and absence of an NRE. In no case was NRE-dependent conversion of the Rel protein to a repressor by cotransfection with DSP1 observed. It is not understood why the current results differ from those reported previously (Brickman, 1999 and references therein).
Sites of the described protein-protein interactions are found in the conserved Rel domains and in the fragment of DSP1 that bears both HMG domains. The Rel domains of p65 and of Dif differ from those of Dorsal and of p50 in that they lack the HMG-domain-interaction site. The HMG domain of DSP1 also interacts with the TATA-binding protein. Similar interactions have been reported for HMG-1 and HMG-2 with the steroid hormone receptors, for HMG-1 with p53, for HMG-1 with HOXD9, and for HMG-2 with Oct2. Thus, the HMG domain may contain a common structural motif for cooperative DNA binding and interaction with other transcription factors. The interaction between TATA-binding protein and DSP1 also seems to be influenced by the glutamine-rich amino-terminal domain in that the full-length DSP1 interacts more avidly with TATA-binding protein than does the HMG-1 domain. These experiments suggest that the amino-terminal glutamine-rich domain may also potentiate the DSP1-Rel protein interaction as well, because all DSP1-Rel interactions seem stronger with full-length DSP1, particularly the weak interactions seen between DSP1 and p65 or Dif, which are observed only with GST-DSP1 and not with GST-DSP1 (178-393) (Brickman, 1999).
Genetic studies have identified numerous sequence-specific transcription factors that control development, yet little is known about their in vivo distribution across animal genomes. This study determined the genome-wide occupancy of the dorsoventral (DV) determinants Dorsal, Twist, and Snail in the Drosophila embryo using chromatin immunoprecipitation coupled with microarray analysis (ChIP-chip). The in vivo binding of these proteins correlate tightly with the limits of known enhancers. This analysis predicts substantially more target genes than previous estimates, and includes Dpp signaling components and anteroposterior (AP) segmentation determinants. Thus, the ChIP-chip data uncover a much larger than expected regulatory network, which integrates diverse patterning processes during development (Zeitlinger, 2007).
ChIP-chip assays were performed with antibodies directed against Dorsal, Twist, or Snail on Toll10b mutant embryos, aged 2-4 h. These embryos contain a constitutively activated form of the Toll receptor, which results in high levels of nuclear Dorsal protein and uniform expression of Twist and Snail throughout the embryo. The high levels of Dorsal, Twist, and Snail cause all cells to form derivatives of the mesoderm at the expense of neurogenic and dorsal ectoderm. Thus, these embryos represent a uniform cell type with respect to DV fate (Zeitlinger, 2007).
The whole-genome ChIP-chip experiments reveal several hundred strong binding clusters of Dorsal, Twist, and Snail with up to 40-fold ChIP enrichment, most of which span regions of ~1 kb in length. To identify the binding patterns of bona fide target enhancers of the Dorsal regulatory network, known enhancers were analyzed. The 22 known enhancers fall into three classes: type 1, type 2, and type 3, based on which levels of nuclear Dorsal regulate their expression (Zeitlinger, 2007).
The 10 type 1 enhancers (associated with twi, sna, miR-1, htl, hbr, mes3, CG12177, ady43A, tin, and Phm) are activated by peak levels of Dorsal in the presumptive mesoderm, and are all constitutively activated in Toll10B mutant embryos. The ChIP-chip experiments identify strong binding peaks (greater than fivefold enrichment) of Dorsal, Twist, and Snail (DTS) within five of the 10 enhancers (twi, sna, miR-1, CG12177 and Phm). Another three enhancers, those associated with htl, tin, and ady43A, show significant but lower (less than fivefold) binding peaks restricted to Twist and Snail (TS) binding. This observation is consistent with earlier studies indicating that these enhancers might be primarily activated by Twist. Hence, eight of the 10 known type 1 enhancers exhibit significant in vivo occupancy by Twist and Snail (Zeitlinger, 2007).
An even greater correspondence between known enhancers and in vivo occupancy is seen for the type 2 [sim, E(spl), vn, rho, vnd and brk] and type 3 enhancers (ths, sog, ind, dpp, zen and tld), which are regulated by intermediate and low levels of the Dorsal gradient, respectively. All 12 enhancers are silenced in Toll10B mutant embryos due to constitutive expression of the Snail repressor. Remarkably, every enhancer exhibits strong DTS or TS peaks with greater than fivefold enrichment in the ChIP-chip assays. Thus, ChIP-chip assays correctly identified 20 of the 22 known Dorsal target enhancers (Zeitlinger, 2007).
Most known DV enhancers are associated with overlapping binding clusters of Dorsal, Twist, and Snail regardless of whether they mediate activation or repression. Moreover, 17 of the 20 binding clusters at known enhancers display greater than fivefold enrichment of Twist and/or Snail. Using these binding criteria, 428 high-confidence DTS regions and 433 high-confidence TS regions were identified across the genome (Zeitlinger, 2007).
To confirm these regions through independent evidence, sequence analysis on these regions was performed using the known consensus binding motifs of Dorsal, Twist, and Snail. As expected, the identified regions are highly enriched in all three binding motifs. Moreover, a large fraction of the motifs is conserved across the 12 sequenced Drosophila species providing evidence that the discovered regions are functionally important. Finally, when motifs that are enriched in these regions were identified de novo, the known binding motifs can be rediscovered. Hence, the regions identified represent putative target gene enhancers of the DV network (Zeitlinger, 2007).
To show that newly identified regions indeed function as enhancers in vivo, putative enhancers were selected of primary DV genes; i.e., those genes that are expressed as localized stripes across the DV axis. In addition to the 22 known DV enhancers, 47 new putative enhancers were identified , some of which appear to regulate the same gene, were identified. By attaching the genomic sequence to a lacZ reporter and expressing the construct in transgenic embryos, seven of these enhancers were shown to be bona fide DV enhancers and that regulation by multiple enhancers occurs (Zeitlinger, 2007).
The wntD gene is expressed in portions of the presumptive mesoderm where it mediates feedback inhibition of Toll signaling. A cluster of DTS-binding peaks was identified in the 5'-flanking region, and the corresponding genomic DNA fragment mediates lacZ expression in the same region of the mesoderm as the endogenous gene. Similar results were obtained with the DTS-binding cluster located in the 5'-flanking region of mes5/mdr49 (Zeitlinger, 2007).
The vnd locus contains a well-documented intronic enhancer that mediates expression in the neurogenic ectoderm and recapitulates the spatial and temporal expression pattern of the endogenous gene. The ChIP-chip analysis detected this enhancer but also revealed two novel clusters further upstream. When tested for lacZ reporter activity, these novel genomic sequences directed lacZ expression in a pattern resembling that of the endogenous gene over different time periods: One directs early vnd expression in the presumptive ventral neurogenic ectoderm (vNE) while the other directs later expression in the medial column (mc) of the developing nervous system. All three enhancers contain evolutionarily conserved binding sites for Dorsal, Twist, and Snail, suggesting that the enhancers are not redundant but may function to fine-tune the vnd expression pattern. Overlapping enhancer activity was also observed for multiple miR-1 enhancers. Overall, as many as a third of all DV genes have multiple binding clusters, and thus might be subject to similar regulatory control (Zeitlinger, 2007).
Several of the occupied regions are associated with Dpp target genes expressed in the dorsal ectoderm. When the tup and pnr intronic sequences are tested in transgenic embryos, both fragments function as authentic enhancers and direct localized expression in the dorsal ectoderm, comparable to the endogenous tup and pnr expression patterns. These results suggest that the Dorsal patterning network directly regulates the expression of Dpp target genes (see below) (Zeitlinger, 2007).
It was noticed that many of the new DTS/TS clusters are associated with AP genes involved in segmentation. Although classical genetic studies argue that AP and DV patterning of the early embryo are controlled by separate maternal genetic programs, it is conceivable that the expression of AP target genes is modulated by the DV network. Indeed, DV modulation of segmentation gene expression has been observed previously (Zeitlinger, 2007).
The gap gene orthodenticle (otd) is expressed in two stripes across the AP axis in the early embryo. The anterior stripe shows diminished expression on the ventral side. Previous studies identified a 5' enhancer that recapitulates the normal expression pattern, including Dorsal-dependent suppression in ventral regions. ChIP-chip identified a strong DTS cluster within the limits of this enhancer. A similar DV bias in the expression pattern was found for the gap gene tailless (tll) and the pair-rule genes runt and hairy. In each case, the regions identified by ChIP-chip overlap or map close to known regulatory regions and contain several Dorsal-binding motifs (Zeitlinger, 2007).
At the gap gene knirps, a DTS-binding cluster was found in a region distinct from the known Bicoid-dependent enhancer. This newly identified genomic region functions as a bona fide enhancer directing expression in the anteroventral domain like endogenous knirps. Thus, the ChIP-chip analysis identified novel AP regulatory regions modulated by DV activity (Zeitlinger, 2007).
In summary, many segmentation genes contain DTS/TS-binding clusters, and at least some of these regions modulate gene expression across the DV axis, particularly in anterior regions of the embryo. It is concluded that the Dorsal gradient does not only regulate primary DV target genes, but rather appears to fine-tune a large number of genes that do not contribute to DV axis formation themselves, at least based on their known genetic function (Zeitlinger, 2007).
Many DTS/TS-binding clusters are also found at genes encoding signal transduction components. Analysis of the network formed by these pathways suggests that the Dorsal gradient controls the expression of many target genes by multiple regulatory pathways (Zeitlinger, 2007).
Dorsal directly represses Dpp expression in the mesoderm and neuroectoderm, leading to localized Dpp signaling in the dorsal ectoderm. Dpp activates a variety of genes, including tup and pnr. Accurate identification of intronic tup and pnr enhancers suggests that these genes are directly regulated by the Snail repressor, in addition to indirect regulation by the Dorsal gradient via Dpp signaling. zen is another well-known target gene of Dorsal in the dorsal ectoderm, and its product, a homeodomain transcription factor, functions synergistically with Dpp signaling. Target genes of Zen also appear to be subject to additional regulation by the Dorsal gradient. In the dorsal ectoderm, Dorsal may regulate gene expression by two mechanisms: direct repression, and indirect repression via Snail (Zeitlinger, 2007).
Similar network configurations regulate gene expression in the neuroectoderm. High levels of Dorsal repress the expression of rho via Snail in the mesoderm, thereby blocking EGF signaling in Toll10b mutant embryos. ChIP-chip data suggest that the Dorsal network regulates additional genes encoding EGF signaling components as well as EGF target genes such as pnt, aop/yan, and argos. In the case of Notch signaling, it is known that the Dorsal network represses Notch target genes such as sim in Toll10B mutant embryos through Snail. The Dorsal network may also regulate Notch signaling more directly, by suppressing genes encoding components of the signaling pathway including Notch itself (Zeitlinger, 2007).
Although repression of neuroectodermal target genes is likely to occur predominantly through Snail, Dorsal also induces the expression of a number of microRNAs in Toll10b mutant embryos, including miR-1. Some of the neuroectodermal genes repressed by Snail are also predicted targets of these microRNAs. Hence, there may be multiple tiers of repression in the DV system, similar to the activities of the gap repressors in the AP system (Zeitlinger, 2007).
In summary, the present ChIP-chip study revealed an unexpectedly broad distribution of binding peaks for Dorsal, Twist, and Snail in the genome, and suggests extensive integration of the Dorsal regulatory network with additional patterning processes, such as Dpp signaling in the dorsal ectoderm and segmentation across the AP axis. In addition to the observed tight correlation between binding peaks and known enhancers, two lines of evidence suggest that a significant fraction of the newly identified regions is functional: First, the bound regions are highly enriched in evolutionarily conserved Dorsal, Twist, and Snail sequence motifs; and, second, several of the identified enhancers were experimentally confirmed by lacZ reporter gene expression in transgenic embryos. Thus, while genetic studies identified core sets of regulators for each developmental process in Drosophila, gene regulation integrates information more widely from several different systems. It is likely that integration of diverse patterning processes will also apply to mammalian development, including stem cell differentiation (Zeitlinger, 2007).
The Dorsal protein has a large N-terminal region of 294 amino acids, homologous to the vertebrate c-rel and its corresponding viral oncogene V-rel, the transforming gene of the reticuloendotheliosis virus strain T (Steward, 1987).
An in vivo structure-function analysis of Dorsal has been performed in order to identify regions of Dorsal that are essential for its homodimerization, nuclear targeting, and interaction with Cactus. All these functions are carried out by regions within the conserved Rel-homology region of Dorsal. The C-terminal divergent half of Dorsal is dispensable for its selective nuclear import. A basic stretch of 6 amino acids at the C terminus of the Rel-homology region is necessary for nuclear localization. This nuclear localization signal is not required for Cactus binding. Removal of the N-terminal 40 amino acids abolishes the nuclear import of Dorsal, uncovering a potentially novel function for this highly conserved region (Govind, 1996).
date revised: 22 January 2000
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